Field of the Invention
The present invention relates to a method for purifying proteins with a phase separation method, by using hydrophobin technology. In particular, the present invention relates to a continuous purification method for hydrophobin fusion protein(s), which results various benefits to different protein industries. In addition, the present invention relates to a method for purifying antibodies directly from a solution with means of a hydrophobin-Protein A fusion and a continuous phase separation technology.
Description of Related Art
Purification of proteins is generally performed by chromatography. Usually gel-chromatographic methods are used, based on ion-exchange, hydrophobic interaction, affinity chromatography and molecular sieving. Methods like electrophoresis and crystallization are also traditional. These methods are well known in the art and suitable for proteins of fairly high market value. In case of bulk protein production these methods, however, are too expensive in order to keep the final product on a compatible price level. Due to similar properties of these proteins several purification steps are usually needed to separate the proteins from each other. This often causes low final yields and therefore a high loss of product.
An example of useful proteins facing problems in purification in a cost-effective way are the commonly used industrial enzymes used as biocatalysts, glycosyl hydrolases, proteases and lipases produced by fungi and bacteria. These are used in e.g. laundry, textile, paper and pulp, food and feed industry. The fact that microbes produce many different enzymes during their growth and the fact that some of these may be undesired in certain applications leads to a need to enrich the active component(s). This enrichment can be performed by choosing appropriate growth conditions, by genetic engineering and/or by downstream processing (e.g. purification of the active component(s)).
Liquid-liquid extraction in an aqueous two-phase system (ATPS) can offer a powerful technique for isolation and purification of proteins. The separation of macromolecules and particles by means of liquid-liquid extraction is well known (Albertsson, 1986, Walter et al., 1985 and Kula, 1990). Mainly polyethylene glycol (PEG)-salt, PEG-dextran and PEG-starch systems have been in use. More recently detergents and detergents with reversed solubility were discovered as suitable methods for separation of macromolecules, and especially for the separation of proteins.
In aqueous two phase systems the desired target e.g. a protein should partition selectively into one phase while the other substances should partition into the other phase. In PEG/salt and PEG/dextran and similar systems there are several driving forces for a substance to phase-partition, for example, van der Waals or electrostatic forces and conformation or ligand interactions (Albertsson, 1986). The factors leading to separation in detergent-based aqueous two-phase systems are suggested to be primarily hydrophobic interactions (Terstappen et al., 1993). Even though a lot of research has been carried out in the field ATPS, none of the designed models provide a holistic view of the phase behavior leaving the predictability of protein separation low (Johansson et al., 1998).
In ATPS the partitioning coefficient K is defined as the concentration (activity in case of an enzyme) of the target in the top phase divided by the concentration (enzyme:activity) of the target protein in the bottom phase. Partitioning coefficients in ATPS systems are usually in the range from less than 1 up to less than 100 (Terstappen et al., 1992 and Terstappen et al., 1993).
  K  =                    c        i            ,      T                      c        i            ,      B      
Yield Y is defined as the amount of target in the top phase divided by the sum of the amount of target in top and bottom. This leads to the following equation:
      Y    T    =      1          1      +              [                                            V              B                                      V              T                                ·                      1            K                          ]            
If the desired substance is directed to the heavier phase (as can be the case when using for example Triton X-114 as the detergent) the yield is defined by the following equation:
      Y    B    =      1          1      +              [                                            V              T                                      V              B                                ·          K                ]            
The volume ratio of the two coexisting phases is defined by the volumes of the lighter over the heavier phase, respectively:
  R  =            V      T              V      B      
Extraction systems based on non-ionic surfactants have been described as an alternative to standard polymer-polymer or polymer-salt systems. Phase forming surfactants are e.g. polyoxyethylene type non-ionic detergents. The basis of this type of aqueous two-phase system is a temperature-dependent reversible hydration of the polar ethyleneoxide head groups. The temperature at which the phase-separation occurs is referred to as the cloud-point temperature (cloud-point extraction). This kind of aqueous two-phase system is especially suited for the extraction of amphiphilic biomolecules. The potential of this type of two-phase system for separating membrane bound proteins from cytosolic and peripheral membrane proteins was first demonstrated by Bordier (1981). In this research a non-ionic temperature sensitive detergent, Triton X-114, was used for separating integral membrane proteins from hydrophilic proteins into a surfactant phase, and the gel electrophoresis results showed that the extraction was successful.
Hydrophobins are bipolar and small proteins, consisting of about 100 to 150 amino acids, of which 8 are cysteine residues. They are expressed by filamentous fungi to help the organism, adapt better to the surrounding environment. The hydrophobins have an affinity for interfaces and are thus known for their ability to form coatings on hydrophobic surfaces. Hydrophobins are usually divided into two classes (class I and class II) based on their hydropathy profiles. Prior art has proposed various uses and applications for hydrophobins and derivatives thereof, such as stabilizing liquid phases (US 20090282729 A1), use as emulsifiers, thickeners and surface-active substances (WO 96/41882), treating materials in cosmetic applications (WO 03/53383) and coating various surfaces (EP 1252516 B1 and WO 03/10331).
Herein the main focus is in protein separation methods that use hydrophobin technology. Relating more closely to the subject matter of the present invention, it has been previously demonstrated, that hydrophobins and hydrophobin fusion proteins can be purified and concentrated with a surfactant based batch phase separation method (U.S. Pat. No. 7,060,669 B1 and U.S. Pat. No. 7,335,492 B2). These US-patents describe isolation and purification of proteins in aqueous two-phase systems (ATPS). Particularly these inventions provide processes and micro-organisms for partitioning of molecules of interest in ATPS by fusing said molecules to target proteins, which have the ability of carrying said molecule into one of the phases.
In U.S. Pat. No. 7,060,669 the targeting protein, such as a hydrophobin-like protein or parts of it, is fused to the product molecule or the component to be separated. First, phase forming materials and eventually possibly also additional salts are added to an aqueous solution containing the fusion molecule or component. The added agents are mixed to facilitate their solubilization. As soon as they are solubilized the two phases are formed either by gravity settling or centrifugation. In the separation the targeting protein drives the product to for instance the detergent-rich phase which could either be the top or the bottom phase. The method is not only useful for purification of products of interest but also for keeping the product or the component of interest, such as a biocatalyst, in a particular phase which enables certain useful biotechnical reactions.
It is however challenging to perform this kind of phase separation in larger scale, because usually if the volumes are high, phase separation is slow. Another problem is the instability of a target protein in required extraction conditions, which causes limitations for utilizing such method. Developing a continuous protein phase separation method would facilitate the handling of high volumes and improve the yields, especially for sensitive target molecules. In a cost-effective protein production process, where production fermenters are working continuously, also the downstream process, herein purification method would preferably be continuous.
Antibodies form an important molecular group especially for pharmaceutical industry and diagnostic industry. They are also the most significant biological medicinal molecular group now and at least in the near future. As the current situation is in most protein industries, also in antibody-relating processes most of the production costs come from purification steps. Prior purification technology is based on sepharose beads, to which antibody-binding proteins have been inserted. For example Protein A column chromatography is commonly used in the pharmaceutical industry for antibody purification. However, it is expensive to produce said beads and their lifetime is limited mostly because of impurities, which cause fouling. Sepharose-based purification and separation of biomolecules are carried out in batch-reactions, which also limits the scalability of these processes.
McLean et al. (2012) have recently studied the production of therapeutic monoclonal antibodies (MA) using genetically modified plants. Researchers used Protein A-oleosin oilbodies (Protein A-OB), expressed in transgenic safflower seeds for capturing MAs, from plants. A purification process for recovering Trastuzumab-antibody was developed, wherein Protein A-OB is mixed with crude extracts from plants engineered to express therapeutic antibodies, the Protein A-OB captures the antibody in the oilbody phase thus leaving the impurities in the aqueous phase. Remaining impurities and purified antibody are finally recovered by the means of centrifugation. Thus, this process also uses phase separation method, but it is aimed at overcoming fouling problems encountered in traditional chromatography technology. In addition, the process is not continuous and uses a different protein approach (oleosin) for recovering therapeutic antibodies.
A continuous purification and concentration method for proteins and antibodies achieved by using phase separation and hydrophobin fusion technologies will raise interest among industries that depend on protein production, such as enzyme, pharmaceutical, food & feed and chemical industries.